Pseudomonas aeruginosa is an important human pathogen that causes infections in a variety of body sites and causes ~8% of CDC-reported healthcare infections. Many P. aeruginosa infections are caused directly by environmental isolates, primarily derived from the potable water system and related fixtures. The low numerical abundance of P. aeruginosa in these environments conflicts with the ease of infection, suggesting there is something fundamentally different about infection from the water system compared to high-dose infection from rich media that are typically used for animal infection models. We predict that the pre-infection niche is a critical component of the natural history of P. aeruginosa infections and that a deeper understanding of this biology will provide novel ideas to decrease exposure risks. Our goal in this proposal is to identify genes required for P. aeruginosa survival in potable water environments and test the impact of potable water and potable-water microbiome members on P. aeruginosa virulence. In data described here, we demonstrate that exposing P. aeruginosa to potable water induces a number of genes involved in pathogenesis and stimulates cytotoxicity in most strains, suggesting that this environment, compared to minimal or rich media, prepares P. aeruginosa for infection. We propose to further investigate P. aeruginosa interaction with the potable water environment via the following Specific Aims: (1) Determine the impact of potable water and potable-water system bacteria on P. aeruginosa virulence and (2) Identify genes important for P. aeruginosa survival in potable water and in association with potable-water system bacteria. Our intent in this exploratory study is to approach the P. aeruginosa pre-infection niche with the same emphasis given to the vector stage of vector-borne pathogens, as the P. aeruginosa pre-infection lifestyle and its role in infection have not been systematically addressed.